Cbfal is essential for the differentiation of osteoblasts from mesenchymal precursors, during early stages of bone formation. It is also required for maintenance of differentiated osteoblasts. Extracellular matrix synthesis stimulates Cbfal activity through a pathway involving MAP kinase and Cbfal phosphorylation. MAP kinase-dependent Cbfal phosphorylation may play an important role in the regulation of bone formation and regeneration. Our long-term goal is to understand how Cbfal phosphorylation is regulated by signals known to affect osteogenesis and the relationship between Cbfal phosphorylation and bone formation and regeneration. Our previous study led to the development of the following two hypotheses that will be in this project: 1. Bone growth factors and cytokines including BMP-2, TGF-bl, IGF-1, FG F-1 and 2, PDGF-BB, and IL-6 regulate MAP pathways and Cbfal phosphorylation. Cbfal phosphorylation is essential for activation of transcription and regulation of osteoblastic proliferation, differentiation and bone formation. 2. Mechanical loading stimulates MAP kinase pathways and Cbfal phosphorylation, thus, increase osteoblastic activity and bone formation. These hypotheses will be addressed through achievement of the following specific aims: 1. Determine the effects including BMP-2, TGF-bl, IGF-1, FGF-1 and 2, PDGF-BB, and IL-6 on MAPK and Cbfal phosphorylation levels; relate these changes to osteoblastic proliferation and differentiation. 2. Determine the effects of mechanical loading on MAPK and Cbfal phosphorylation levels as well as on osteoblastic proliferation and differentiation. These studies will provide useful fundamental new knowledge regarding the molecular mechanism of bone formation and regeneration by bone growth factors and cytokines as well as by mechanical loading.